Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of CD4+CD25+Foxp3+ regulatory T cells (Treg). Our recent studies have focused on defining the mechanism of action of the CD4+CD25+Foxp3+ Treg cells in vitro and on an analysis of their potential dysfunction in autoimmune disease: (1) The suppressive capacity of Treg on T cell activation has been well documented. However, little is know concerning the effects of Treg on other cell types that are involved in immune responses. By co-culturing pre-activated CD4+CD25+ T cells with B cells in the presence of polyclonal B cell activators, we found that B cell proliferation was significantly suppressed. The suppression of B cell proliferation was due to increased cell death caused by the CD4+CD25+ T cells in a cell-contact dependent manner and depends on the up-regulation of granzyme B in the CD4+CD25+ T cells. Activated Treg cells preferentially killed antigen presenting, but not bystander B cells. This result raises the possibility that one site of action of regulatory T cells in mediating suppression in vivo is the APC. Because only highly activated, TCR-stimulated Treg can kill B cells, there appears to be greater regulation of this aspect of their function compared to their capacity to suppress T-cell activation by acting directly on responding T cells. Such a control mechanism may facilitate a rapid response of B cells to pathogen-derived thymic independent antigens and thus avoid suppression by Treg cells during the early phases of an immune response. After activation and expansion in vivo following multiple rounds of antigen stimulation, B-cell apoptosis induced by Treg cells may result in a decrease not only in autoantibody production but, more importantly, in a decrease in the antigen-presenting function of activated B cells. (2) Recent studies have defined naturally occurring Treg as expressing the forkhead family transcription factor, Foxp3. Although Foxp3 is necessary for the development of Treg in the thymus, it may not be sufficient. Engagement of CD28 is critical for the generation of Treg, but IL-2 does not appear to be required in vivo. The role of IL-2 in the maintenance and function of Treg in the periphery still remains to be clarified. Recent reports have demonstrated that peripheral CD4+Foxp3- T cells can be induced in vitro to express Foxp3 in the presence of TGF-beta. These induced cells, termed iTregs, are functionally similar to thymic-derived Tregs in that they are anergic, suppressive, and capable of inhibiting disease in vivo. We have examined the cytokine and costimulatory molecule requirements for the TGF-beta-mediated induction and maintenance of Foxp3 by CD4+CD25- Foxp3- cells. Although signalling via CD28 is not required, IL-2 plays a critical role in the induction, but not the maintenance of Foxp3 expression in vitro and in vivo. One major question that remains unresolved by these studies is whether and under what conditions iTreg are generated in vivo The therapeutic potential of TGF-beta iTreg also remains to be determined. Our results indicate that T cells that have been recently activated in vitro under Th1, Th2, or non-polarizing conditions cannot be induced to express Foxp3 under the conditions we have used for naive cells. It is unclear whether autoantigen-specific memory cells that might be isolated from patients will also be difficult to convert to iTreg expressing Foxp3. (3) Recent studies have revealed the presence of CD4+CD25+Foxp3+ Tregs in human peripheral blood, where they constitute up to 5% of the CD4 T cells. These cells are similar to those described in the mouse in that they require cell-to-cell contact to exert their suppressive effects. Tumor necrosis factor (TNF) is a pleiotropic cytokine critical for cell trafficking, inflammation, maintenance of secondary lymphoid organ structure, and host defense against various pathogens. TNF plays a critical role in bridging innate and adaptive immunity. However, its role in regulating the function of Tregs or their impact on effector cells is presently unknown. Rheumatoid arthritis (RA) is one of the most common human autoimmune diseases, with a prevalence of nearly 1%. Treatment of RA patients with TNF blockers results in significant clinical benefit. Recent studies in RA patients have suggested that the function of Tregs may be impaired. Treatment of RA patients with anti-TNF antibodies appeared to result in an increased frequency of Tregs and reversed their defect in inhibition of cytokine secretion. The cellular targets and the molecular mechanism of TNF action on those cells have not been delineated. We have demonstrated that human Tregs express TNFRII and that the percentage of Tregs that express TNFRII can be enhanced by exposure to TNF. The addition of TNF or agonistic antibody to TNFRII reversed the suppressive activity of the human Tregs by down modulating the expression of FOXP3. The beneficial effect of TNF blockade in autoimmune/ inflammatory diseases could involve the restoration of immune homeostasis by permitting the full expression of Treg function. Strategies designed to manipulate TNF signaling in Tregs may result in novel therapeutic approaches to augment the limited and/or inadequate function of these regulatory T cells in inflammatory or autoimmune diseases. (4) It is widely accepted that thymic-derived Foxp3+ Treg must be activated via their TCR to exert their suppressive effects. However, this result raises the question of how the very small number of antigen-specific Treg in the polyclonal populations used to inhibit autoimmune disease can be so efficient in their suppressive effects in vivo. it remains possible that Treg might directly or indirectly exert potent and permanent effects on effector T cells rendering them refractory or anergic to stimulation via the TCR. To test this hypothesis we co-cultured Foxp3+ Treg with effectors and determined whether they became anergic and/or suppressive. Two distinct populations of cells were isolated from co-cultures. About 1/3 of the responders had divided, while 2/3 remained in a resting state. Most importantly, both cell populations were completely unresponsive to re-stimulation via the TCR in the presence of APC. The population of undivided cells also failed to respond to exogenous IL-2. Co-culture with Foxp3+ Treg also induced suppressor function in the responders., The induced Treg did not express any detectable Foxp3 mRNA or intracellular Foxp3 protein while Foxp3 is readily detected at high levels in conventional Treg. The suppressive activity of the induced Treg is never as potent as that observed with an equivalent number of Foxp3+ Treg. In contrast to our failure to neutralize suppression in the co-cultures of Foxp3+ Treg and responders with anti-IL-10 or anti-TGF-beta, we did observe moderate neutralization of the suppressive activity of induced Treg with anti-TGF-beta, but not anti-IL-10. Our results are compatible with the view that some of the potent effects of thymic-derived Foxp3+ Treg in the suppression of immune responses in vivo may be secondary to conversion of potentially autoreactive effector cells to anergic non-pathogenic cells or to induction of cells with the capacity to actively suppress.